The overall goal of this project is to develop a molecular framework for the family of membrane bound O-acyltransferases (MBOATs) using the bacterial DltB protein as a model system. Acylation has emerged as an abundant and biologically significant protein modification in biology, with important implications for therapy. The MBOATs are one family of acyltransferases that are conserved from bacteria to man and add a long chain fatty acid molecule to the oxygen atom of a metabolite or a serine and threonine side chain of a protein. These polytopic integral membrane proteins play important metabolic roles and several human members such as diacylglycerol acyltransferase 1 (DGAT1), ghrelin O-acyltransferase (GOAT) and hedgehog acyltransferase (HHAT) have emerged as important drug targets in metabolic diseases and cancer. The bacterial MBOAT protein, DltB, participates in the biosynthesis of a major component of gram-positive bacterial cell wall and therefor represents a drug target for gram-positive bacterial pathogenesis. The lack of molecular information on MBOAT proteins is due to the difficulty in preparing these proteins in recombinant form for biochemical and structural analysis. In preliminary data, we have overcome these difficulties to prepare recombinant DGAT1, GOAT and DltB proteins and have developed biochemical assays for them. Most recently, we have prepared a DltB sample that is suitable for biochemical analysis and structure determination using X-ray crystallography. This places us in a unique position to use the DltB protein as a model system to make important breakthroughs in understanding the structure and function of MBOAT proteins. The specific Aims of this proposal are to (1) Determine the atomic resolution structure of the DltB MBOAT protein using X-ray crystallography, and (2) Establish structure-function correlations with in vitr and in vivo DltB activity assays to evaluate DltB mutants. Together, these studies will provide important new insights into the structure and function of the DltB MBOAT protein and a scaffold for developing novel probes and inhibitors to combat bacterial pathogenesis. These studies will also have implications for understanding the structure-function of the greater MBOAT family and pave the way to address their substrate specificities and to develop novel MBOAT-specific probes and inhibitors for therapy.